JP7198264B2 - Methods for storing biopharmaceuticals - Google Patents

Description

FIELD OF THE INVENTION This invention relates generally to biopharmaceuticals ; more particularly, to improved methods for preserving biopharmaceuticals using conventional lyophilizers.

The field related to biopharmaceutical preservation technology has advanced in recent years, and includes "Preservation by Evaporative Drying" as disclosed in U.S. Pat. No. 9,469,835, issued October 18, 2016. Significant improvements have been achieved with the advent of certain superior methods known as PBV.

Currently, conventional lyophilizers (freeze dryers) are not designed to perform preservation of biopharmaceuticals and other compositions using PBV protocols.

Additionally, conventional freeze-drying protocols often expose biopharmaceutical compositions to extremely low temperatures that tend to affect the activity of these compositions.

Since many laboratories and industrial facilities possess conventional freeze dryers, biopharmaceutical compositions are typically outdated such as conventional freeze-drying, which results in lower product activity and stability. stored using the protocol of

A need exists for improved methods for storing biopharmaceuticals using conventional lyophilizers.

More particularly, there is a need for improved methods that incorporate the use of conventional lyophilizers for preserving biopharmaceuticals according to PBV technology.

Additionally, there is a need to maintain aseptic isolation of biopharmaceutical compositions when storing biopharmaceuticals according to these methods.

Special preservative formulations will be required to protect biopharmaceutical compositions during PBV protocols.

The present disclosure relates to methods for implementing PBV protocols for preserving biopharmaceuticals using conventional lyophilizers.

The present disclosure also relates to steps for maintaining separation of biopharmaceuticals to achieve aseptic drying using conventional lyophilizers.

Further, the present disclosure complies with good manufacturing practice (GMP) to achieve aseptic drying of biopharmaceutical compositions using conventional freeze dryers that are disposed outside of clean room areas. on how to.

Finally, the present disclosure relates to methods and formulations for the production of ambient temperature stable micronized biopharmaceuticals suitable for mucosal or transdermal delivery to a patient.

These and other solutions to the problem will be understood by those of ordinary skill in the art upon a thorough review of the accompanying description, drawings, and claims.

Preservation by Vaporization is a preferred technique in the field of biopharmaceutical preservation technology. This is because, during the initial drying, also called the "primary drying" step, the step in which most of the water is removed from the preservation composition, the process uses simultaneous water to achieve a relatively mild environment. This is because boiling, sublimation of ice crystals, and evaporation are used. This gentle environment is particularly gentle to sensitive proteins, viruses, bacteria, and other cellular entities, and is applied for the stabilization of vaccines, blood, and microbiome components at ambient temperature. there is a possibility. Subsequent continued drying (also known as "secondary drying") slightly increases the glass transition temperature of the preserved composition, resulting in a mechanically stable amorphous dry vitreous foam. resulting, wherein the biopharmaceutical encapsulated within the foam is protected and protected against (i) storage beyond 90 days (“long-term storage”) and (ii) sometimes subsequent reconstitution or delivery to a patient. saved for The disclosed method allows one skilled in the art to store biopharmaceuticals according to PBV protocols adapted for practice using conventional lyophilizers. Therefore, specialized equipment is not required to store biopharmaceutical compositions using PBV technology.

A biopharmaceutical composition preserved using PBV technology provides superior efficacy compared to the same composition preserved using conventional freeze-drying technology. The extreme temperatures experienced using conventional freeze-drying techniques tend to destroy the protein epitopes and useful components of these compositions, e.g. to give the product.

GMP production of biopharmaceutical compositions, including but not limited to vaccines, can be achieved using the methods disclosed herein. Thus, the method provides improved, more efficient, GMP-compliant processing of materials for stabilization for industrial applications.

Certain preservation formulations and protocols are disclosed to protect biopharmaceuticals during storage and to prevent material devitrification.

Other advantageous effects will be recognized by those skilled in the art upon a thorough review of this specification.

A method for storing biopharmaceuticals is presented. Figure 2 shows a results chart for an experiment testing the activity of PBV LAIV after micronization and subsequent storage. 1 shows a DSC plot associated with an experiment detailed herein. FIG. 4 shows a DSC plot associated with another experiment, detailed herein. FIG. 4 shows a DSC plot associated with another experiment, detailed herein.

For purposes of illustration and not limitation, details and descriptions of certain preferred embodiments are provided below to enable those skilled in the art to make and use the invention in its various aspects and embodiments. . However, these details and descriptions are merely representative of certain preferred embodiments, and myriad other embodiments not explicitly described will be readily apparent to those of ordinary skill in the art upon a thorough review of this disclosure. The Rukoto. Therefore, any reader of this disclosure should interpret that the scope of the invention by the claims is not intended to be limited by the embodiments described and illustrated herein. be.

We now disclose, in general embodiments, methods for preserving biopharmaceuticals . This method includes the following steps in order:
Step (i): providing in a container an aqueous storage composition, the storage composition comprising one or more biopharmaceuticals ;
Step (ii): Place the container and the stored composition therein on a temperature-controlled shelf in the vacuum chamber of a freeze dryer (wherein the temperature-controlled shelf provides a shelf temperature and the vacuum chamber is adapted to provide a certain vacuum pressure therein);
Step (iii): Lowering the shelf temperature to below 0° C. and reducing the vacuum pressure to below 1.0 torr, thereby converting the storage composition to a two-phase slush state (wherein the storage composition is ice crystals in a two-phase slush state);
Step (iv): raising the shelf temperature above 0° C. and maintaining the vacuum pressure below 1.0 torr, thereby converting the storage composition into a mechanically stable vitreous foam; and step (v): increasing the shelf temperature above 40° C. to increase the glass transition temperature of the mechanically stable vitreous foam.

For the purposes of this specification, the term "preservation composition" generally preserves biopharmaceuticals during long-term storage at ambient temperatures from -30°C to +40°C and subsequent reconstitution or delivery to a patient. means a composition for The preservation composition generally comprises one or more sugars and one or more biopharmaceuticals , where the biopharmaceuticals are one or more bacteria, one or more viruses, one or more proteins, or combinations thereof. A storage composition is aqueous in its initial state, ie, a liquid or gel containing some percentage composition of water above 1 percent. In this initial state, various ingredients such as biopharmaceuticals , sugars, sugar alcohols, and other ingredients are selected and mixed together. However, during the first drying step of the PBV protocol, also known as "primary drying", water is removed from this aqueous composition under vacuum pressure by simultaneous boiling, evaporation and sublimation. Thus, the preservation composition will be substantially dehydrated after primary drying and will generally be an amorphous dry glassy matrix or foam (herein referred to as "mechanically stable vitreous foam").

Thus, further for the purposes of this specification, the term "mechanically stable vitreous foam" refers to a storage composition after PBV primary drying (wherein the storage composition is substantially free of water and structural forming an amorphous, dry vitreous foam with sufficient mechanical strength to maintain its shape). Specifically, during PBV storage, as soon as the storage composition begins to boil, gas bubbles nucleate and grow inside the composition, which migrate (stabilize) early in the process. In time, the viscosity of the composition as a result of dehydration prevents the water vapor bubbles from growing further, and near the end of primary drying or when most of the water has evaporated, the bubbles Fixed within the composition, the foam becomes mechanically stable.

In one embodiment, during step (iv), the shelf temperature is raised above 20° C. and the vacuum pressure is lowered below 0.3 Torr in the vacuum chamber of the freeze dryer.

In another embodiment, during step (iv), the shelf temperature is raised to above 20° C. and the vacuum pressure is lowered to less than 0.3 Torr in the vacuum chamber for a first period of time; And after the first period of time, the shelf temperature is raised above 30° C. and the vacuum pressure is maintained below 0.3 Torr in the vacuum chamber.

In some embodiments, the shelf temperature is raised above 50° C. during step (v).

In various embodiments, the aqueous storage composition (before primary drying) comprises a liquid or gel.

Gels can be produced by chemical cross-linking between polymers contained in the storage solution, or between polymers and other molecules or ions. Ions can include cations, ie Ca++, or anions.

Gels can be produced by chemical cross-linking during heating of a mixture of frozen droplets containing another cross-linking component. Frozen droplets can be produced using cryo-palletization, eg, spraying the solution into a cryogenic liquid such as liquid nitrogen (LN2).

A gel can also be produced by cooling a storage composition that turns into a gel below 0°C.

In some embodiments, cooling of a polymer solution that can form a gel during cooling is performed by cryo-pelletization. Cold pelletization is a process in which material is dispersed into many droplets before cooling.

While many formulations are possible, preservation compositions generally include one or more non-reducing sugars, one or more sugar alcohols, or combinations thereof.

In some embodiments, at least one of said one or more non-reducing sugars is selected from the group consisting of sucrose, trehalose, raffinose, methylglucoside, and 2-deoxyglucose. However, other non-reducing sugars known to those skilled in the art can be incorporated as well.

In some embodiments, at least one of said one or more sugar alcohols is selected from the group consisting of mannitol, isomalt, sorbitol, and xylitol. However, other non-reducing sugars known to those skilled in the art can be incorporated as well.

A preservation composition can include one or more cellular cryoprotectants, one or more extracellular cryoprotectants, or a combination thereof.

In some embodiments, at least one of the one or more cell cryoprotectants is selected from the group consisting of glycerol, propylene glycol, erythritol, sorbitol, mannitol, methylglucoside, and polyethylene glycol.

In some embodiments, at least one of the one or more extracellular cryoprotectants is glutamic acid, glycine, proline, serine, threonine, valine, arginine, alanine, lysine, cysteine, polyvinylpyrrolidone, polyethylene glycol, and selected from the group consisting of hydroxyethyl starch;

The storage composition may further comprise one or more antifoam agents.

In some embodiments, at least one of the one or more antifoam agents is selected from the group consisting of polypropylene glycol and ethylene glycol.

In certain embodiments, the method comprises, prior to step (i), loading the biopharmaceutical with one or more permeable cell cryoprotectants , followed by with a preservative solution to form the preservative composition.

One or more biopharmaceuticals include bacteria, viruses, proteins, or combinations thereof.

In one embodiment, one or more biopharmaceuticals comprises a combination of one or more bacteria, one or more viruses, and one or more proteins.

In various embodiments, the container can comprise one of a serum vial, plastic bottle, metal container, or tray.

In some embodiments, the container can include a porous membrane having a pore size of 0.25 μm or less, and the storage composition can be separated from the environment of the vacuum chamber by the porous membrane. Porous membranes can include any filter used for sterilization of aqueous solutions.

In some embodiments, the container is also sealed within a sterile pouch.

In some embodiments, the container can contain a plurality of metal or ceramic spheres provided within a volume of the container along with the storage composition.

In some embodiments, the method comprises step (v) followed by the metal contained therein in said container to convert the mechanically stable vitreous foam into particles/powder. or using ceramic spheres to grind the mechanically stable vitreous foam (“ball-milling”).

In some embodiments, particularly if the biopharmaceutical is intended for mucosal or transdermal delivery, the storage composition can comprise at least 5 parts disaccharide to 1 part sugar alcohol. Additionally, the storage composition can further comprise a water-soluble amino acid or salt thereof. In some embodiments, water-soluble amino acids or salts thereof can include arginine, glutamic acid, glycine, proline, or combinations thereof.

In another aspect, a method for aseptic production of a thermostable biopharmaceutical is disclosed. This method includes the following steps:

Within a first area of the clean room: placing a storage composition, the storage composition comprising one or more biopharmaceuticals , in a container; sealing the opening of the container with a porous membrane; forming a membrane-sealed container (wherein the porous membrane comprises a pore size that is 0.25 microns or less); and placing the membrane-sealed container in a sterilized medical grade sterilization pouch. , heat sealing the sterilization pouch;

transporting the sterilization pouch and the membrane-sealed container therein from a first area of the cleanroom to a second area, wherein the method comprises transporting the sterile pouch and the membrane-sealed container therein; on a temperature-controlled shelf within the vacuum chamber of a lyophilizer; and by adjusting the vacuum pressure and shelf temperature a drying protocol, wherein the drying protocol renders the storage composition mechanically stable. converting to a vitreous foam);

Following performing the drying protocol, removing the membrane-sealed container from the sterilization pouch and then returning the membrane-sealed container to the first area of the cleanroom (here, the first area of the cleanroom); Within the area of , this method sterilizes the porous membrane to ensure that the mechanically stable vitreous foam within the container remains isolated from external moisture during subsequent storage. replacing the cup or covering the filter with a water impermeable sterile sticker; optionally converting the mechanically stable vitreous foam in the container to a powder).

A clean room is typically designed to include three areas, often referred to as A, B, and C. A is the first, maximally sterile part of the clean room. According to FDA regulations, an individual may perform any work outside of area A (within areas B and C) of the cleanroom and may be present if covered with clean clothing and/or is also possible. Individuals are not allowed to enter area A, which is the cleanest. To perform minor manipulations such as delivery of bottles or instruments into area A, or to open and close containers, or the like, only hands covered with sterile gloves should be placed in area A for short periods of time. It is allowed to cross the line that separates the B region. Tools or bottles should be isolated from the environment outside area A before moving into area A. For example, utensils or bottles should be placed in a sterile pouch (which will be removed prior to placing utensils or bottles in area A) to ensure that area A remains sterile. be.

In various embodiments, the dry foam results from the nucleation and growth of vapor bubbles during boiling, appears as a foamy foam formed during drying of a liquid, or a drying foam containing gaseous inclusions. It can appear as a hydrogel.

These and other embodiments are devised and supported according to the following experimental examples:

Example 1: Storage by Evaporative Drying (PBV)
Storage by evaporative drying (PBV) is a relatively new technique for stabilization of fragile vaccines and other biopharmaceuticals at ambient temperature (AT). PBV implementation requires simultaneous control of temperature and vacuum pressure. This could be done by contacting the drying chamber with a vacuum source and providing heat to the sample being dried to counteract the cooling resulting from water evaporation. The heat source can be electromagnetic heating or conventional heat flow from a heated shelf on which the sample is placed during drying. There can be many designs of vacuum drying equipment that can carry out the PBV process. PBV can also be carried out using conventional lyophilizers, such as those currently used for the production of dried biologics using freeze-drying protocols. Since the practice of freeze-drying typically requires the use of relatively high vacuums, conventional freeze dryers require pressures in excess of 1 Torr or even 2 Torr inside the drying chamber. not designed to automatically control the vacuum pressure when the This limits the range of PBV processes that can be performed automatically using conventional freeze dryers. However, various embodiments herein describe how a conventional lyophilizer can be used to store biopharmaceuticals at ambient temperature and without operator involvement. How to program the drying protocol for automatic execution of non-intrusive PBV protocols, which will be required for GMP production and FDA approval as well as approval of various regulatory agencies outside the United States. It is disclosed that it is possible to

According to PBV technology, primary drying should be performed from a partially frozen slush state. Therefore, to perform PBV, a given preservation composition, such as a preservation mixture (PM) of a biological suspension and preservation solution (PS), or a hydrogel containing a biologic and PS should first be partially frozen. "Frozen" means that there are ice crystals formed inside the sample. "Partially frozen" means that the sample temperature is at least 10°C above Tg'. Here, Tg' is the temperature below which the storage composition is in a solid state with no liquid phase remaining and comprising crystallized solids and amorphous solids (glass). One important difference between PBV and conventional freeze-drying is that during primary freeze-drying (or lyophilization) the temperature of the sample should be at or below Tg'. However, the sample temperature during the PBV primary drying step should be at least 10°C above Tg' to ensure the presence of a liquid phase between ice crystals. Also, to ensure that this liquid not only evaporates but also boils, the vacuum pressure in the chamber should be below the equilibrium water vapor pressure above the liquid. Here, consistent with accepted terminology, primary drying is defined as the initial drying step during which the sample loses approximately 90% of the initial amount of water in the sample.

Prior to PBV storage, biopharmaceuticals should be mixed with a preservation solution (PS) designed to protect the biopharmaceutical from the harmful effects of freezing and drying. Appropriate selection of PS is important to ensure successful storage. The lower the freezing temperature, the more important it becomes to include conventional cryoprotectants in PS. It is important to use both cellular cryoprotectants that penetrate inside the cellular and extracellular cryoprotectants that protect cell and viral membranes and envelopes.

Example 2: Animal Cell Storage CTV-1 is a human T-cell leukemia cell line. CTV-1 cells were grown in a 37° C., 5% CO2 incubator and suspended in RPMI medium (85-90%) and heat-inactivated FBS (10-15%) with penicillin-streptomycin. cultured in the condition Cells were split every 4-5 days and maintained at around 1E6 cells/mL. Cells were then harvested and concentrated by centrifugation to approximately 0.5-1E7 cells/mL.

Procedure: 5.5 g of cell culture was mixed with 0.5 ml of 50% glycerol and loaded at room temperature (RT) for 30 minutes. Loaded cells were treated with double volumes of two stock solutions: PS1 containing 30% sucrose, 15% MAG; Two stock mixtures (PM) were prepared by mixing with PS2 containing PVP). PMs were placed in 5 ml serum vials (0.5 ml per vial) and dried inside a freeze dryer using the PBV protocol described below.

The PBV protocol used included the following steps:
partially freezing the PM by lowering the shelf temperature to -18°C and holding at -18°C for 18 minutes;
reducing the vacuum pressure inside the drying chamber to 0.1 Torr and maintaining this pressure for 5 minutes to ensure that the PM is frozen;
Increase the shelf temperature to 0° C. and the vacuum pressure to 0.9 Torr and hold this pressure and shelf temperature for 50 minutes;
Raise the shelf temperature to 10° C., reduce the vacuum pressure to 0.5 torr, and hold this pressure and shelf temperature for 22 minutes;
Raise the shelf temperature to 10° C., reduce the vacuum pressure to 0.5 torr, and hold this pressure and shelf temperature for 22 minutes;
Raise the shelf temperature to 20° C., reduce the vacuum pressure to 0.1 torr, and hold this pressure and shelf temperature for 22 minutes;
Raise shelf temperature to 30° C., maintain vacuum pressure at 0.1 Torr, and maintain this pressure and shelf temperature for 20 hours;
Raise shelf temperature to 40° C., maintain vacuum pressure at 0.1 Torr, and maintain this pressure and shelf temperature for 20 hours.

PBV samples were reconstituted with 0.5 ml of RPMI culture medium after storage and further diluted 1:2 with RPMI culture medium. Cell viability was tested using an Invitrogen Tali™ image-based cytometer with the Tali™ Viability Kit-Dead Cell Red, a solution of propidium iodide. Propidium iodide is impermeable to living cells, but will enter dead cells (cells with compromised membranes) and fluoresce red when bound to nucleic acids. This Viability Kit contains the protocol used for the measurements: (https://tools.thermofisher.com/content/sfs/manuals/mp10786.pdf). A Tali™ cytometer was used to monitor cell health/viability using the propidium iodide protocol, calculate the percentage of live/dead cells, and measure average cell size. Measurements on this instrument were performed with a propidium iodide fluorescence (PIF) threshold set at 440 RFU and a restricted average cell size cutoff of 4-20 μm.

Results: Cell survival after drying with PS1 was less than 10%; cell survival after drying with PS2 was greater than 75%. Thus, PVP provided cell membrane protection during storage.

Example 3 Storage of Bacterial Cells A suspension of bacterial cells was mixed 3:1 with 9% glycerol (in PBS). Three PMs were then prepared by mixing this suspension with twice the volume of the three stock solutions:
(PS1): 7.86% glutamic acid, 10% sucrose, 5% mannitol, 2% NaOH, and 4.76% PVP (adjusted to pH=7);
(PS2): 7.86% glutamic acid, 10% sucrose, 5% mannitol, 2% NaOH (adjusted to pH=7); and (PS3): 7.86% glutamic acid, 10% sucrose, 5% mannitol, 2 NaOH. %, and PEG 4.76% (adjusted to pH=7).

Procedure: PMs were placed in 5 ml serum vials (0.5 ml per vial) and dried inside a lyophilizer using the PBV protocol described below.

The PBV protocol used included the following steps:
partially freezing the PM by lowering the shelf temperature to -18°C and holding at -18°C for 18 minutes;
reducing the vacuum pressure inside the drying chamber to 0.1 Torr and maintaining this pressure for 5 minutes to ensure that the PM is frozen;
Increase the shelf temperature to 0° C. and the vacuum pressure to 0.9 Torr and hold this pressure and shelf temperature for 50 minutes;
Raise the shelf temperature to 10° C., reduce the vacuum pressure to 0.5 torr, and hold this pressure and shelf temperature for 22 minutes;
Raise the shelf temperature to 10° C., reduce the vacuum pressure to 0.5 torr, and hold this pressure and shelf temperature for 22 minutes;
Raise the shelf temperature to 20° C., reduce the vacuum pressure to 0.1 torr, and hold this pressure and shelf temperature for 22 minutes;
Raise shelf temperature to 30° C., maintain vacuum pressure at 0.1 torr, and maintain this pressure and shelf temperature for 20 hours.

Results: After PBV drying, the material was reconstituted with 0.5 ml PBS after storage and plated on BHI agar after serial dilution. Plates were stored in a 37°C incubator for 24 hours. With PS3, bacterial survival after drying is less than 30%; with PS2, bacterial survival after drying is less than 60%; with PS1, bacterial survival after drying is greater than 90%. Met. Therefore, PVP provided additional protection to bacteria during storage.

Example 4: Process Validation and Scale-Up Drying in Serum Vials This study was conducted to verify the reproducibility of the PBV drying process. Human albumin was used as a model protein in these experiments. A 2 ml stock mixture containing 20 mg recombinant human albumin (Novozymes ALBiX), 266 mg sucrose and 133 mg isomalt was filled into a 20 ml serum vial, placed on a steel tray and placed in a Virtis freeze dryer. I put it on the shelf. The tray was completely filled with a total of 120 vials. The PBV foam drying protocol consisted of a 3 hour primary drying at 1500 millitorr at temperatures ranging from -15°C to 30°C and a 20 hour secondary drying at 50°C. The appearance of the dry foam (a mechanically stable vitreous foam) appeared very uniform across the vial with no visible splashing.

Glass Transition Temperature (Tg) Measurement Objective: To measure the Tg of PBV albumin samples taken from different areas of the drying tray to test for uniform drying conditions in the freeze dryer.

Experimental Procedure: Five sample vials of PBV albumin were collected from the four corners of a full drying tray (20 sample vials total). Three DSC sample pans were prepared in the drying room using the dried material from each vial. Each sample contained approximately 5 mg of hand-milled PBV foam. The sample pan was run on a DSC 8000 (Perkin Elmer, Massachusetts, USA) equipped with an autosampler. Each pan was scanned from -50°C to 80°C at a rate of 20°C/min and the Tg was calculated using Pyris data analysis software (see example calculation below).

result:

Water Activity Measurement Objective: To measure water activity (aw) from PBV albumin samples taken from different areas of the drying tray to test for uniform drying conditions in the lyophilizer.

Experimental Procedure: Five sample vials of PBV albumin were collected from the four corners of a full drying tray (20 sample vials total). The PBV foam in each vial was manually ground into a powder. Approximately 150 mg of powder from each vial was tested on an Aqualab 4TE Dewpoint Water Activity Meter (Decagon Devices, Washington, USA) placed in a dry chamber.

result:

Example 5: Drying inside a 1 liter plastic bottle covered with a 0.2 μm sterilizing filter (membrane) and separated from the chamber environment.
As in the previous example, PM was prepared and filled into 1 liter plastic containers at 150 g/container. After PM preparation, each container was covered with a 0.2 μm filter used for sterilization of aqueous solutions.

The shelf temperature is reduced to −18° C., after which the vacuum pressure is reduced to less than 0.1 Torr and remains equal to 0.1 Torr until the end of the process. The shelf temperature was then increased to 30°C and held at 30°C for 24 hours. The shelf temperature was then increased to 40°C and held at 40°C for 24 hours.

Example 6: Preservation of Live Attenuated Influenza Vaccine (LAIV) A single high titer stock of frozen LAIV strain (A/17/Texas/2012/30(H3N2)) was prepared using the preservation by evaporation (PBV) technique. saved using . For storage, LAIV was thawed and mixed 1:1 with various storage solutions (PS) with pH 7 to form storage mixtures (PM). PMs were dispensed into serum vials and dried using the PBV process with secondary drying temperatures of 45°C and 50°C. Various vaccine preparations were tested for activity using a modified LAIV TCID50 assay protocol provided by the CDC.

Experiment Flu-1:
In experiment Flu-1 we used 7 different PSs including:
PS1: 30% sucrose, 10% methyl glucoside (MAG), 0.5% gelatin;
PS2: 30% sucrose, 1140% methyl glucoside (MAG), 0.5% albumin;
PS3: 30% sucrose, 10% mannitol;
PS4: 30% sucrose, 10% mannitol, 0.5% gelatin;
PS5: 30% sucrose, 10% acetoglucose;
PS6: 30% sucrose, 10% acetoglucose, 0.5% gelatin; and PS7: 30% sucrose, 10% MAG.

Unfortunately, PS6 crystallized out during drying and was not further studied. We hypothesize that solution PS6 was supersaturated and gelatin somehow affected the nucleation of acetoglucose crystals.

Results: The activity (log ₁₀ of TCID ₅₀ /ml) of the dried vaccine after PBV and after 1.6 years of storage at room temperature (RT) is shown in the table below.

Experiment Flu-2:
In experiment Flu-2, we used PS containing a lower concentration of mannitol (33% sucrose + 7% mannitol) and a secondary drying temperature of 50°C.

This experiment was performed while we were initially setting up and optimizing our unique TCID50 protocol, and for this reason we decided that storage at RT The activity of the preserved vaccine was first measured after 5 months after establishing a reliable quantitative measurement. Vaccine titers (2E7 TCID50/ml) showed only a 0.62 log drop in activity relative to frozen controls after PBV drying and 5 months of room temperature storage. A sample from this preparation was used in the micronization studies below.

Experiment Flu-3
In experiment Flu-3, we tested several novel formulations, including one that further reduced mannitol concentration and maintained a higher secondary drying temperature of 50°C (35% sucrose + 5% mannitol). investigated.

Results: The activity (TCID ₅₀ /ml) of FLU-3 formulations after storage at room temperature (RT) and 37°C is shown in the table below.

Conclusion: PBV drying of LAIV using a 35% sucrose and 5% mannitol formulation under a process that includes a secondary drying step at 50°C allows the production of thermostable LAIV.

Example 7: Ball milling of PBV dry placebo formulations to formulate heat stable LAIV in dry powder form with a particle range suitable for nasal delivery using ball milling techniques. A placebo sample was used to study ball milling of PBV formulations. Placebo samples were prepared and evaluated as follows:
(i) To prepare placebo PMs, 100 g of sterile cell culture medium was mixed with 100 g of PS containing 33% sucrose and 7% mannitol;
(ii) PMs were aliquoted into 20 mL serum vials (2 mL/vial) prior to drying;
(iii) the material was subjected to the same PBV protocol used to dry the vaccine in earlier experiments, with a 1-day secondary drying at 45°C;
(iv) After drying, the PBV placebo foam was first crushed inside the serum vial with a sterile spatula to break up the PBV foam and reduce the particle size to a few hundred microns;
(v) at a frequency of 15 Hz (which we have previously determined to be a suitable frequency for obtaining particles of about 20 microns) for a period of 45, 75, or 90 minutes; Subsequent micronization was performed using a LabWizz Laboratory-Scale Ball Mill;
(vi) The powder was passed through a copper sieve with a mesh size of 63 μm to remove any agglomerates or larger particles. The portion of powdered material that would not have passed through the sieve was noted. The mesh size was chosen to be 63 μm as this size is designated for sieving LactoHale lactose powder used as a dry powder bulking agent in the preparation of dry LAIV vaccines for respiratory delivery. . We have also found that the particles that can pass through this mesh are substantially smaller than 63 μm and are well suited for respiratory delivery; Suspended in mineral oil and evaluated using conventional microscopy.

The inventors have discovered that ball milling of PBV foam to a particle size suitable for respiratory delivery can be accomplished using a rocking frequency of 15 Hz and milling times ranging from 45 to 90 minutes ( (see table below). Shorter milling times tend to result in more variable particle sizes and an increased proportion of particles that do not pass through the 63 μm sieve. The inventors concluded that milling for 75 minutes at a rocking frequency of 15 Hz was the optimal protocol to produce micronized vaccine powders for respiratory delivery.

The results are shown in FIG.

Activity of PBV LAIV after Micronization and Subsequent Storage evaluated the impact of Vaccine micronization was performed as described above for placebo samples.

Results: Activity per milligram (TCID50) of dry PBV LAIV powder micronized at 15 Hz for 45, 75, and 90 minutes followed by storage at 4°C, RT, and 37°C for 1.5 months. are shown below.

Ball milling resulted in low activity loss and the micronized vaccine remained stable for at least 1.5 months at RT. However, the inventors found a significant decrease in micronized vaccine stability during storage at 37° C. compared to non-micronized vaccine preparations. This may be due to crystallization of mannitol or sucrose from supersaturated sucrose/mannitol glasses. This crystallization may be initiated by the nucleation of mannitol crystals on the surface of cracks that occur during grinding.

Example 8: Effect of Micronization on Live Attenuated Rotavirus Vaccine (LARV) Preserved by PBV:
The LARV vaccine was mixed 1:1 with PS containing 30% sucrose and 10% mannitol and preserved by PBV. Similar to the Flu vaccines discussed so far, particle size reduction was performed in two steps: manual grinding followed by ball milling (micronization) using a Laarmann Labwizz laboratory size ball mill. First, a vial that had been stored at room temperature for 3 months was opened and the dried foam material inside the vial was manually ground using a spatula in a dry room with a relative humidity of 15-18%. Approximately 120 mg of manually crushed foam was recovered from each vial. The particle size after manual grinding was typically several hundred microns. The manually ground foam was then placed into 2 mL metal containers each containing a 3 x 2 mm steel ball. The container was placed inside a ball mill and vibrated at a rate of 15 Hz for 30 minutes to reduce the particle size to approximately 50 microns. A portion of this powder was dried in vacuum at 45° C. for 1 day.

We found very small (<0.2 logs) loss of viral activity after micronization and subsequent storage at RT for 5 months. However, after 5 months of storage at 37° C., it underwent a 0.8 log loss of activity that was not observed for the non-micronised formulation. This finding correlates with the reduced stability of PBV flu vaccines after micronization as described above.

Our determination of Tg, using a Differential Scanning Calorimeter, indicates that the decrease in viral activity at 37°C in the micronized sample is due to crystals nucleating on the surface of the fraction. supported the working hypothesis that it may be related to the sugar crystallization of

We investigated a PBV formulation prepared with PS containing 30% sucrose and 10% mannitol. For each formulation, we prepared three independent samples hermetically sealed inside aluminum pans. We did not see any unexpected results with the non-micronized formulations and we found Tg from −50 to 80° C. during the first, second and third DSC runs. could be measured. See FIG. 3, which shows a DSC scan of Rota-1 PM4 powder (hand-milled, not micronized, 3 runs).

However, only for micronized powders, the first run of each sample pan had observable glass transition behavior, after which we found that the DSC associated with crystallization during warming A rapid drop in output was clearly observed. This phenomenon, also known as devitrification, is often associated with the formation of metastable crystals above the glass transition temperature if the low viscosity does not stop the growth of crystals that often form on the surface of the fraction in the glassy state. observed in supersaturated mixtures. Figure 4 shows a DSC run for a dry micronized PM4 formulation.

Devitrification is an irreversible kinetic crystallization process in supersaturated solutions that did not crystallize during cooling, thus forming a stable glass below the Tg. Crystal nuclei form at temperatures below that at which crystals can grow, or crystal nucleation can occur only on the surfaces of cracks in the glassy state, so crystals are typically cooled during cooling. transformation does not occur. If the viscosity of the solution decreases during warming above the Tg, the crystal nuclei that form at lower temperatures begin to grow at a rate that increases with increasing temperature - irreversibly transforming the material in the crystallized state. convert. The higher the Tg, the higher the temperature at which devitrification occurs for a given heating rate.

We also measured the Tg in a sample of the micronized dry formulation that was dried for an additional day at 45°C in vacuum. No effect of devitrification during warming from the glassy state was detected in these samples. A DSC run for the dry micronized PM4 formulation after additional drying at 45° C. is shown in FIG.

After additional drying of the dry micronized formulation, Tg measurements were significantly higher than those obtained for the micronized formulation without the additional drying step (Tg was slightly above 30°C). (Tg was slightly above 50°C). This explains why no devitrification was observed in the further dried powder.

Devitrification does not necessarily occur in all vitrification mixtures and depends on the composition of the mixture. According to our preliminary findings, this is more pronounced in mixtures containing mannitol compared to mixtures containing MAG. It is believed herein that an increase in Tg will reduce or eliminate devitrification.

The claimed invention is applicable to the pharmaceutical industry, particularly the vaccine industry, but is also applicable to other parts of the pharmaceutical industry.
References
US Patent No. 9,469,835, issued October 18, 2016.
Pushkar,NSand VLBronshtein.1988.The mechanism of ice formation on rewarming of the frozen cryoprotectant solutions.Proc.Ukrainian Acad.Sci.10:68-70.

Claims (15)

A method for storing a biopharmaceutical comprising:
(i) providing in a container an aqueous storage composition, the storage composition comprising one or more biopharmaceuticals ;
(ii) placing the container and the storage composition therein on a temperature-controlled shelf within the vacuum chamber of a lyophilizer, wherein the temperature-controlled shelf is adapted to provide a shelf temperature; and the vacuum chamber is adapted to provide a vacuum pressure therein;
(iii) a freezing protocol comprising:
The shelf temperature is reduced to less than 0° C. , the vacuum pressure is reduced to less than 1.0 torr, and the temperature of the storage composition is maintained above Tg', thereby rendering the storage composition biphasic. performing a freezing protocol comprising converting to a slush state, wherein said preservation composition comprises ice crystals and an aqueous liquid phase in a two-phase slush state;
(iv) a primary drying protocol comprising:
The shelf temperature is raised above 0° C. and the vacuum pressure is maintained below 1.0 torr, thereby boiling water from the storage composition in the two-phase slush condition to mechanically performing the primary drying protocol comprising forming a stable vitreous foam;
(v) a secondary drying protocol comprising:
A method comprising, in turn, performing said secondary drying protocol comprising increasing said shelf temperature above 40°C to increase the glass transition temperature of said mechanically stable vitreous foam. during step (iv), the shelf temperature is raised above 20° C. and the vacuum pressure is lowered below 0.3 torr within the vacuum chamber for a first period of time; 2. The method of claim 1, wherein after a period of 1, the shelf temperature is raised to greater than 30[deg.]C and the vacuum pressure is maintained at less than 0.3 torr in the vacuum chamber. 3. The method of any one of claims 1 or 2, wherein the aqueous storage composition comprises a liquid or gel. The preservation composition comprises one or more non-reducing sugars, one or more sugar alcohols, or combinations thereof, and at least one of the one or more non-reducing sugars is sucrose, trehalose, raffinose. , methylglucoside, and 2-deoxyglucose, and at least one of said one or more sugar alcohols is selected from the group consisting of mannitol, isomalt, sorbitol, and xylitol. 4. The method according to any one of 1 to 3. The preservation composition comprises one or more cellular cryoprotectants, one or more extracellular cryoprotectants, or combinations thereof, and at least one of the one or more cellular cryoprotectants is , glycerol, propylene glycol, erythritol, sorbitol, mannitol, methylglucoside, and polyethylene glycol, and at least one of said one or more extracellular cryoprotectants is glutamic acid, glycine, proline, serine , threonine, valine, arginine, alanine, lysine, cysteine, polyvinylpyrrolidone, polyethylene glycol, and hydroxyethyl starch. Claims 1-, wherein said storage composition comprises one or more antifoaming agents, and at least one of said one or more antifoaming agents is selected from the group consisting of polypropylene glycol and ethylene glycol. 6. The method of any one of 5. loading the biopharmaceutical with one or more cell cryoprotectants prior to step (i), followed by mixing the loaded biopharmaceutical with a storage solution to form the storage composition; 2. The method of claim 1, further comprising forming. 8. The method of any one of claims 1-7, wherein said one or more biopharmaceuticals comprise bacteria, viruses, and proteins, or combinations thereof. The method of any one of claims 1-8, wherein the container is one of a serum vial, a plastic bottle, a metal container, or a tray. 10. The container according to any one of claims 1 to 9, wherein the container comprises a porous membrane having a pore size of 0.25 μm or less, and the storage composition is separated from the environment of the vacuum chamber by the porous membrane. Method. The method of any one of claims 1-10, wherein the container is sealed within a sterile pouch. A method according to any preceding claim, wherein a plurality of metal or ceramic spheres are provided within the volume of the container. 13. The method of claim 12, further comprising crushing a mechanically stable vitreous foam within the vessel using metal or ceramic spheres contained therein following step (v). described method. 14. The method of any one of claims 1-13, wherein the preservation composition comprises at least 5 parts disaccharide to 1 part sugar alcohol. 15. Any of claims 1-14, wherein the preservation composition further comprises a water-soluble amino acid or salt thereof, and wherein the water-soluble amino acid or salt thereof comprises arginine, glutamic acid, glycine, proline, or a combination thereof. or the method described in paragraph 1.

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